Adult nerves of the mammalian central nervous system (CNS) show poor regenerative ability after axonal injury. Spontaneous growth of injured axons does occur, but ceases after a few hundred microns without traversing the site of the injury and elongating in the distal stump. The failure to regenerate has been attributed to the inhospitable nature of the nerve's environmental milieu, including the inability of astrocytes (the scar forming cells) to support growth, the paucity of macrophages and/or their products, and the formation of mature oligodendrocytes which inhibit axonal growth.
The non-neuronal cells contribute to the CNS environment and they have been implicated in the failure of CNS regeneration in mammals. These cells include astrocytes, which hypertrophy to form fibrous scars in response to lesion and oligodendroglia, which are inhibitory to axonal growth. The astrocytic scar is largely composed of type-1 astrocytes and has been considered to prevent growth by forming a physical barrier. The time course of scar formation is long, however, and it seems unlikely that a barrier formed by the scar would prevent regenerative axonal growth in the immediate post traumatic period. Type-1 astrocytes in rat optic nerve have been shown to express laminin, a molecule implicated in support of axonal outgrowth, prenatally, during the development of the optic nerve. Mammalian adult brain type-1 astrocytes normally do not express laminin, except only transiently following an injury to the brain. In contrast, laminin is continuously expressed in regenerative fish optic nerve. In in vitro preparations, axons grow in close contact with type-1 astrocytes. Mature oligodendrocytes are now believed to be non permissive for axonal growth. Growing axons will avoid contacting mature oligodendrocytes, in vitro. During development, most axonal growth in the optic nerve takes place before birth, before any oligodendrocytes have differentiated. It seems, therefore, that axonal regeneration in mammals is hindered by both the presence of mature oligodendrocytes, which are non permissive to axonal growth, and the type-1 reactive astrocytes, which are lacking the supportive element(s), in contrast to the fish optic nerve.
Prior work of the present inventors and others has shown that the CNS of lower vertebrates, specifically regenerating fish optic nerve, is a source of factors which, when applied at the appropriate time and in appropriate amounts to injured mammalian adult optic nerves, can support regenerative axonal growth (Schwartz et al., 1985; Hadani et al., 1984; Lavie et al., 1987; Cohen et al., 1989; Lavie et al., 1990; and European Patent No. 172987). An activity cytotoxic to oligodendrocytes was attributed to substances within these preparations, which presumably enable the fish optic nerve to overcome the inhibitory activity associated with oligodendrocytes (Sivron et al., 1990 and 1991). The cytotoxicity in vitro was shown to be not only to fish oligodendrocytes, but also to rat oligodendrocytes (Published European Application EP No. 415,321, Sivron, T. et al., 1990 and Cohen et al., 1990). These substances are associated, directly or indirectly, with macrophages or other blood derived cells.
Interleukin-2 (IL-2) is a lymphokine known to be synthesized and secreted by T cells after activation with antigen or mitogen in the presence of IL-1 (Smith , 1988). IL-2 in the immune system has been considered to be an important cytokine, responsible for either inhibition or progression of many immune responses (Liang et al., 1989). In contrast, very little is known about the role of IL-2 in the brain. In the nervous system, some of the observations related to the effects of IL-2 on oligodendrocytes appear to be contradictory. Recent studies have attributed an inhibitory effect on oligodendrocytes to the cytokine IL-2 (Saneto et al., 1986 and 1987), while other studies have shown a proliferative effect of IL-2 on oligodendrocytes (Benveniste and Merril, 1986). IL-2 in mammals has been shown to be a product of lymphocytes (Smith, 1988) and some reports have indicated that fish lymphocytes may have IL-2-like activity (Capsi and Avtalion, 1984).
An association between IL-2 and injury in the CNS in general, and in the brain in particular, has also been pointed out. Nieto-Sampedro et al., 1987, found IL-2 activity after brain injury. Similarly, Liang et al., 1989, found IL-2 in brain lesions created by MPP+(1-methyl-4-phenyl pyrimidine). In addition, up-regulation of IL-2 binding sites was observed by Araujo et al., 1989, in rat hippocampus as a result of injury. Nevertheless, no association between IL-2 and CNS regeneration has yet been suggested.
Other recent studies have suggested that regeneration might be prompted by treatments that circumvent growth hindrance by oligodendrocytes, e.g., applications of factor cytotoxic to oligodendrocytes such as tumor necrosis factor (TNF) (U.S. Pat. No. 5,580,555, and Schwartz et al., 1991) or of antibodies directed against the oligodendrocyte-associated inhibitors (Schnell et al., 1990).
Robbins et al., 1987, have reported that stimulation of rat astrocytes in vitro resulted in the generation of a cytotoxic factor that is functionally similar to TNF. They also reported that recombinant human TNF (rhTNF) has cytotoxic activity directed against rat oligodendrocytes. Selmaj et al., 1988, reported on the testing of rhTNF for its effect on myelinated cultures of mouse spinal cord tissue. They found that rhTNF induced delayed-onset oligodendrocyte necrosis and a type of myelin dilatation.
Despite substantial research efforts worldwide, no safe and effective means for causing regenerative growth of CNS axons in mammals, and particularly humans, has yet been developed. Such a means, and particularly a pharmaceutical which can be injected at the site of desired regeneration would be very desirable in order to help alleviate post-traumatic paraplegia or quadriplegia, blindness, deafness, surgically associated axotomy, etc.